Efficient gene editing in induced pluripotent stem cells enabled by an inducible adenine base editor with tunable expression

The preferred method for disease modeling using induced pluripotent stem cells (iPSCs) is to generate isogenic cell lines by correcting or introducing pathogenic mutations. Base editing enables the precise installation of point mutations at specific genomic locations without the need for deleterious double-strand breaks used in the CRISPR-Cas9 gene editing methods. We created a bulk population of iPSCs that homogeneously express ABE8e adenine base editor enzyme under a doxycycline-inducible expression system at the AAVS1 safe harbor locus. These cells enabled fast, efficient and inducible gene editing at targeted genomic regions, eliminating the need for single-cell cloning and screening to identify those with homozygous mutations. We could achieve multiplex genomic editing by creating homozygous mutations in very high efficiencies at four independent genomic loci simultaneously in AAVS1-iABE8e iPSCs, which is highly challenging with previously described methods. The inducible ABE8e expression system allows editing of the genes of interest within a specific time window, enabling temporal control of gene editing to study the cell or lineage-specific functions of genes and their molecular pathways. In summary, the inducible ABE8e system provides a fast, efficient and versatile gene-editing tool for disease modeling and functional genomic studies.

Editing with modified synthetic gRNAs for KLF1 Y290H (A), KLF1 L300P (B) and CDAN1 F360L (C).AAVS1-iABE8e iPSCs were electroporated with the three gRNAs separately after pre-treating the cells with 0.5µg/ml doxycycline for 48 hours.Editing was quantified in duplicates (1 and 2) by Sanger sequencing and analysed by EditR.NGS was performed in one of the duplicates and analysed by CRISPResso.The targeted base change is marked 'Target' and any by-stander adenine conversions in the editing window are marked as 'BS'.KLF1 Y290H gRNA had no BS conversion, KLF1 L300P bad one bystander (BS) and CDAN1 F360L had two bystander conversions (BS1 and BS2).The gRNA binding region of the target gene was considered for the total A to G edit quantification.
Supplemental Figure .S4. Sequential adenine base editing in AAVS1-iABE8e iPSCs using synthetic sgRNAs.Synthetic modified gRNAs to create KLF1 Y290H, KLF1 L300P and CDAN1 F360L mutations by sequential electroporation of the gRNAs into AAVS1-iABE8e iPSCs.The cells were electroporated with the three gRNAs sequentially after pre-treating the cells with 0.5µg/ml doxycycline for 48 hours.Editing was quantified in duplicates (1 and 2) by Sanger sequencing and analysed by EditR.NGS was performed in one of the duplicates and analysed by CRISPResso.The targeted base change is marked as 'Target' and any bystander adenine conversions in the editing window are marked as 'BS'.The KLF1 Y290H gRNA did not result in any BS conversion, while KLF1 L300P had one BS conversion and CDAN1 F360L had two BS conversions (BS1 and BS2).The gRNA binding region of the target gene was considered for the total A to G edit quantification.
Supplemental Figure .S5. Simultaneous adenine base editing in AAVS1-iABE8e iPSCs using synthetic sgRNAs.Synthetic modified gRNAs to create four mutations, KLF1 Y290H, KLF1 L300P, CDAN1 F360L and CDAN1 T884A, were electroporated simultaneously into AAVS1-iABE8e iPSCs after pre-treating the cells with 0.5µg/ml doxycycline for 48 hours to induce ABE8e expression.Editing was quantified in duplicates (1 and 2) by Sanger sequencing and analysed by EditR.NGS was performed in one of the duplicates and analysed by CRISPResso.The targeted base change is marked as 'Target', and any by-stander adenine conversions in the editing window are marked as 'BS'.The KLF1 Y290H gRNA did not result in any BS conversion, while KLF1 L300P and CDAN1 T884A had one BS conversion each, and CDAN1 F360L had two BS conversions (BS1 and BS2).The sequence reads within the gRNA binding region were considered for total edit quantification.To detect of integration of iABE8e casette

RT-qPCR primers
To detect expression of ABE8e Note: # CACCG in the forward oligo and AAAC in the 5' end and C in the 3' end of the reverse oligo are overhangs for cloning into pLKO5.sgRNA.EFS.GFP vector.
* TACACGACGCTCTTCCGATCT and AGACGTGTGCTCTTCCGATCT are tag sequences in the forward and reverse primers, respectively, for the primary PCR for NGS.
$ AATGATACGGCGACCACCGAGATCTACAC and CAAGCAGAAGACGGCATACGAGA are tag sequences in the forward and reverse primers, respectively, used for the secondary PCR for NGS.NNNNNNNN represent different index sequences in the forward and reverse primers for for multiplexing.
Supplemental Figure.S1.Characterization of AAVS1-iABE8e iPSCs.(A) Results of junction PCRs using a forward primer that binds upstream of the DSB site and a reverse primer binding to the exogenous Tet-On-ABE8e cassette (left junction) and a forward primer binding to the Tet-On-ABE8e cassette and reverse primer binding downstream of the DSB site (right junction).(B) Karyotyping analysis, revealing no numerical or structural chromosomal abnormalities in the AAVS1-iABE8e iPSCs.(C) Immuno-staining of pluripotency markers NANOG.SSEA4, Oct4 and Tra-1-60 in AAVS1-iABE8e iPSCs.Scale bar: 250µm.(D) Trilineage differentiation of AAVS1-iABE8e iPSCs confirmed by Immuno-staining of the expression of PAX6 (Ectoderm), SOX17 (Endoderm) and Brachyury (Mesoderm).Nuclei (Blue) were stained with DAPI.Scale bar: 250µm.Supplemental Figure.S2.Design and analysis of gRNAs used for generating single and multiple mutations in AAVS1-iABE8e iPSCs.gRNAs targeting KLF1 Y290H, KLF1 L300P, CDAN1 F360L and CDAN1 T884A were manually designed and analyzed by BE-Hive for the predicted adenine base conversion efficiencies.The editing windows of the gRNAs are marked by a blue box.The targeted base change is marked 'Target' and the by-stander conversions in the editing window are marked as 'BS1' and 'BS2'.The predicted efficiencies of each conversion and the combinations of conversions are tabulated.The gRNA binding region of the target gene was considered for the total A to G edit quantification.Supplemental Figure.S3.Single locus adenine base editing in AAVS1-iABE8e iPSCs.(A-C) Figure S1A.(B) Full-length immunoblots for Figure 1C.(*lanes not shown in the main figure).
To detect CDAN1 F360L mutation by Sanger sequencing and NGS To detect KLF1 Y290H and KLF1 L300P mutation by Sanger sequencing and NGS To detect FANCA L1082P mutation by Sanger sequencing and NGS To dectect mutaions in the HBG1 and HBG2 promoter sites by NGS To detect expression of b-Actin NGS Secondary PCR primers $

Table S1 :
List of oligos used in this study